The discovery of circulating cell-free fetal DNA in maternal plasma [Lo Y M et al. (1997), Lancet, 350: 485-487] has catalyzed a series of new avenues for non-invasive prenatal diagnosis, including fetal RhD blood group genotyping [Lo Y M et al. (1998), N Engl J Med, 339: 1734-1738, Finning K et al. (2008), BMJ, 336: 816-818], fetal sex determination for sex-linked disorders [Costa J M, Benachi A, Gautier E (2002), N Engl J Med, 346: 1502], chromosomal aneuploidy detection [Lo Y M et al. (2007), Proc Natl Acad Sci USA. 104: 13116-13121; Chiu R W et al. (2008), Proc Natl Acad Sci USA 105: 20458-20463; Chiu R W, Cantor C R, Lo Y M (2009), Trends Genet, 25: 324-331; Fan H C et al. (2008), Proc Natl Acad Sci USA, 105: 16266-16271; Chiu R W et al. (2011), BMJ 342: c7401; Yu S C et al. (2014), Proc Natl Acad Sci USA, 111: 8583-8588], and detection of monogenic diseases [Lo Y M D et al. (2010), Sci Transl Med, 2: 61ra91; Lam K W et al. (2012), Clin Chem.; New M I et al. (2014), The Journal of Clinical Endocrinology & Metabolism, 99: E1022-E1030; Yoo S-K et al. (2015), Clinical Chemistry; Ma D et al. (2014), Gene, 544: 252-258; Tsui N B et al. (2011), Blood, 117: 3684-3691].
Among the aforementioned applications, accurate deduction of the fetal DNA fraction (also known as fractional fetal DNA concentration or fetal DNA percentage) is important for accurate statistical interpretations of the results of non-invasive prenatal diagnosis through the use of plasma DNA, especially when the statistics models dependent on this parameter are used in the detection of chromosomal aneuploidies [Sparks A B et al., (2012), Am J Obstet Gynecol, 206: 319 e311-319 and the determination of monogenic disease inheritance [Lo Y M et al. (2007), Proc Natl Acad Sci USA. 104: 13116-13121; Lo Y M D et al. (2010), Sci Trans/Med, 2: 61ra91; Lam K W et al. (2012), Clin Chem.; New M I et al. (2014), The Journal of Clinical Endocrinology & Metabolism, 99: E1022-E1030; Yoo S-K et al. (2015), Clinical Chemistry; Tsui N B et al. (2011), Blood, 117: 3684-3691]. For instance, the fetal DNA fraction is a central parameter in Relative Haplotype Dosage Analysis (RHDO) used to accurately determine which maternal haplotype was transmitted to fetus [Lo Y M D et al. (2010), Sci Transl Med, 2: 61ra91; Lam K W et al. (2012), Clin Chem.; New M I et al. (2014), The Journal of Clinical Endocrinology & Metabolism, 99: E1022-E1030]. In this diagnostic approach, the underlying principle is that the relative dosage of the maternal haplotype transmitted to the fetus will be slightly over-represented than the untransmitted one and the fetal DNA fraction is used to determine statistical significance of the over-representation.
To date, there are already many methods developed to estimate the fractional fetal DNA concentration in the maternal plasma of a pregnant woman. For example, the specific signal originating from Y chromosome is used to deduce the fetal DNA fractions in pregnancies carrying male fetuses [Chiu R W et al. (2011), BMJ 342: c7401; Lo Y M et al. (1998), Am J Hum Genet, 62: 768-775; Lun F M et al. (2008), Clin Chem 54: 1664-1672; Hudecova I et al. (2014), Plos One, 9: e88484]. However, the Y chromosome specific signal based approaches are not applicable for pregnancies carrying female fetuses. An alternative approach is to use single nucleotide polymorphism (SNP), such that the ratio of the fetal specific alleles to the shared alleles is calculated to infer the fetal DNA fraction. In this approach, genotype information has to be known and should fit one of following situations: (a) the mother is homozygous while the fetus is heterozygous; (b) both paternal and maternal genotypes are homozygous but with different alleles [Lo Y M D et al. (2010), Sci Transl Med, 2: 61ra91; Liao G J et al. (2011), Clin Chem, 57: 92-101. However, on one hand, in the actual clinical scenarios during non-invasive prenatal diagnosis, the fetal genotypes are not available beforehand. On the other hand, the prevalence of paternal discrepancy can be as high as 30%, suggested by an epidemiological study on paternal discrepancy around the world [Bellis M A, Hughes K, Hughes S, Ashton J R (2005) J Epidemiol Community Health, 59: 749-754], which limits the availability of the paternal DNA for data analysis. Even though a parental-genotype-independent algorithm was developed to obviate the prerequisite of extra genotype information by utilizing the high-depth sequencing of maternal plasma DNA (for example targeted sequencing of maternal plasma DNA) across different SNP sites [Jiang P et al. (2012), Bioinformatics, 28: 2883-2890], additional effort is required to capture a set of regions of interest, for example, through the use of hybridization- or amplicon-based enrichment systems [Sparks A B et al., (2012), Am J Obstet Gynecol, 206: 319 e311-319; Liao G J et al. (2011), Clin Chem, 57: 92-101].
In addition to SNP dependent approaches, SNP-independent methods are also being explored. For example, fragment sizes of maternal plasma DNA can be used for estimating fetal DNA fraction [Yu S C et al. (2014), Proc Natl Acad Sci USA, 111: 8583-8588; Kim S K et al. (2015), Prenatal diagnosis: n/a-n/a] because the fetal-derived DNA are generally shorter than maternal-derived DNA [Lo Y M D et al. (2010), Sci Transl Med, 2: 61ra91]. However, some other conditions will influence the accuracy of the size-based fetal DNA fraction estimation, for example, systemic lupus erythematosus [Chan R W et al. (2014), Proc Natl Acad Sci USA, 111: E5302-5311]. As an alternative, fetal-specific epigenetic changes, such as methylated RASSF1A and unmethylated SERPINB5 sequences, were demonstrated to be fetal markers for fetal DNA fraction prediction irrespective of genotype information [Chan K C et al. (2006), Clin Chem, 52: 2211-2218; Chim S S et al. (2005), Proc Natl Acad Sci USA, 102: 14753-14758]. However, the analytical procedures for quantifying these epigenetic markers involve either bisulfite conversion or digestion with methylation-sensitive restriction enzymes, and may thus potentially affect the precision of these methods.
Accordingly, it is desirable for new techniques to provide fetal DNA fraction information from maternal plasma.